Voltage-gated Na+ channels play a central role in controlling excitability in the heart and serve as receptors for local anesthetic antiarrhythmic drugs. it is of considerable interest to cardiovascular biologists and clinicians who treat patients with cardiac arrhythmias to consider how ion channels work. There are two sets of essential features of ion channel function; the ability to open and close in response to a biological stimulus (gating), and the ability to permit ions to flow at high rates through the pore with precise selectivity (permeation). The long-term goal of this proposal is to understand, at the molecular level, ion permeation through voltage-gated Na+ channels. Significant insights can be gained from biophysical analysis of site-directed mutants of the channel protein. The cardiac isoform of the Na+ channel is sensitive to blockade by group IIB(Cd2+/Zn2+) divalent cations and insensitive to block by quanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX). The block by divalent cations is mediated by a cysteine (cys) residue which is present in the pore of cardiac but not skeletal muscle of nerve Na+ channels. Such differences in isoform phenotype will be exploited to map the pore of the channel by sequential replacement of amino acid in the permeation pathway with cys and examination of Cd2+/Zn2+ and toxin blockade. These experiments will allow us to define which amino acid residues contribute to the pore and will provide information regarding the quaternary structure and symmetry of this region of the protein. We will also investigate the mechanism of the subconductance state produced by Zn2+ block of the TTX- insensitive channel variants. Acidic residues in the permeation pathway also influence permeation and blockade. We will study the mechanism by which these negatively charged residues influence permeation by neutralizing them and examining the conduction properties over a range of ionic strengths and permeant ion concentrations. Finally we will investigate local anesthetic blockade with a view to localizing the site(s) of drug-channel interaction. At least one site of blockade by these drugs is in the pore; we will isolate this mechanism by using membrane-impermeant changed local anesthetics in combination with alteration of candidate residues in the pore. The combination of recombinant DNA techniques and high-resolution electrical recording promises to further our understanding of the structure-function relationship of the channel pore and address questions relating to ion transport and channel blockade on a more mechanistic level.